Ion exchange tumor targeting (IETT)

ABSTRACT

A compound for local, i.e. intratumoral or intracavitary administration, for targeting tumors comprising dextran, the charge of which has been modified through covalent binding of charged groups, such as charged amino acids. The compound further comprises a functional group for treating or imaging the tumor.

REFERENCE TO RELATED APPLICATIONS

The present application is the national stage under 35 U.S.C. 371 ofinternational application PCT/SE99/01837, filed Oct. 12, 1999 whichdesignated the United States, and which international application waspublished under PCT Article 21(2) in the English language.

BACKGROUND OF THE INVENTION

Tumor specific targeting, is an intensive research field withinoncology. The rationale is to reduce side effects and increase the drugdose that can be delivered to the cancer patient thus enhancing thetherapeutic efficacy.

Examples of such targeting are antibody oriented targeting, i.e.immuno-targeting, and controlled drug delivery systems using variouspolymers as drug carriers (1, 2, 3, 4, 5, 6).

Tabata et al (11) describes intravenous administration of dextran withnegative charges. This gives very low specific accumulation of thedextran to the tumor. The specific accumulation or the target tonon-target ratio (ttn-ratio) is never exceeding 10 (shown in FIGS. 8 and9, page 219).

Syrigos et al (12) describes intravesical administration ofradiolabelled tumor-associated monoclonal antibody giving a specificaccumulation or target to non-target ratio (ttn-ratio) ranging between 2and 6.

SUMMARY OF THE INVENTION

This invention relates to compounds that, when administered locally,intratumorally or intracavitarily, show unexpectedly high accumulationto tumors and that can deliver various functional groups, including boththerapeutic or imaging agents to these sites, to mediate tumor therapyor imaging.

The inventor has discovered that due to the ionic properties of tumors,polymers with an appropriate charge can be selectively targeted totumors through electrostatic interactions in an ion exchange likereaction. The charged character of tumors could be explained by tumorshaving enlarged blood vessel surface, abnormal interstitium, and highcontent of sialic-acid at the cell surface. All these contain charged,mainly anionic, carbohydrates (sulphated carbohydrates) and proteins(collagen) (7, 8). The selective accumulation is unexpectedly high whenadministered locally, intratumorally or intracavitarily.

By using these charged groups characteristic for tumors as target, theinvention avoids the general limitations of tumor specific targeting,such as low affinities between targeting compound and tumor etc.Targeting tumors with compounds according to the invention avoids theproblem of tumor heterogeneity, i.e. the limitation of antibody-basedcancer therapies that target only a single type of tumor cells in atumor containing multiple cell types. Further more, tumor therapy orimaging using compounds according to present invention do not require aninitial pre-targeting step, as many prior art methods do.

The compounds according to the invention show, when administeredlocally, intratumorally or intracavitarily, unexpectedly high affinitiesfor tumors, i.e. they have an unexpectedly high target to non-target(ttn) ratio (see Example 2). The charge of the compounds can be adaptedto the charge of the tumor to be treated, thereby increasing the targetto non-target ratio of the compound to the tumor. When looking at thegeneral properties of solid tumors, it seems probable that the principleof the invention, i.e. selective accumulation in tumor tissue throughinteractions between the charges of the tumor and the polymer, isfunctional in most human solid tumors.

In preferred embodiments, the compounds of the invention comprise atargeting compound, which is a charged polymer that binds selectively tothe tumor through electrostatic interactions. The targeting compound ismodified to include a functional group, which may be part of the polymeror attached to the polymer as a tag. Where the compound is to be usedtherapeutically the functional group is a toxin, a drug, a radioactivemolecule or a precursor thereof. Where the compound is to be used fortumor imaging rather than therapy, the functional group may also be adetectable label, such as a radioactive molecule.

DETAILED DESCRIPTION OF THE INVENTION

The compounds according to the invention are for local, i.e.intracavitary or intratumoral administration. Examples of tumorssuitable for such administration are tumors growing in body-cavitiessuch as urinary bladder tumors, ovarian tumors, certain brain tumors,and for intratumoral administration, pancreatic tumors, and head andneck tumors. When the compounds according to the invention areadministered locally, intratumorally or intracavitarily the specificaccumulation or ttn-ratio ranges between 700 and 2050, which far exceedsspecific accumulation when administered according to prior art.

The compounds according to the invention comprise a charged polymer,which causes the targeting, and a functional group, which causes thetherapy or imaging.

In preferred embodiments the targeting polymer of the compound accordingto the invention is a poly-alcohol, such as dextran. When using dextran,a preferred molecular weight is between 10³-10⁶ Dalton. In a preferredembodiment the charged side groups of the polymer are amino acids havingcharged side chains, or derivatives thereof, such as ornithine, lysine,arginine, histidine, glutamic acid, aspartic acid etc. The amino acidsare coupled to dextran by activating the hydroxyl groups of dextranthrough partial oxidation to aldehydes which react with the free aminogroups of the amino acids. This procedure is described in Foster, R L.1975 (10). An example of a resulting compound (lysine-dextran) accordingto the invention is shown in FIG. 1. In a preferred embodiment an aminoacid is coupled to between 15-30% of the glucose residues of dextran.

The resulting charge of the compound can be positive, negative orneutral depending of the requirements, i.e. the electrostatic propertiesof the type of tumor to be targeted.

Examples of the functional group of the compound according to theinvention include drugs (e.g. antibiotics, anti-virals, anti-fungals),toxins (e.g. ricin), radio-nuclides (e.g. Cu-64, Cu-67, Sr-89, Y-90,Tc-99m, I-131, Sm-153, Ho-166, Re-186, Re-188, Bi-212), hormoneantagonists (e.g. tamoxifen), heavy metal complexes (e.g. cisplatin),oligo-nucleotides (e.g. antisense oligo-nucleotides), chemotherapeuticnucleotides, peptides, non-specific (non-antibody) proteins, boroncontaining compounds (e.g. carborane), photodynamic agents (e.g.rhodamine), enediynes (e.g. calichesmicins), and transcriptions basedpharmaceuticals.

Coupling of the functional group could be done either directly to thepolymer or through a bifunctional chelate, such as an aminatedradio-metal chelate. Direct coupling of the functional group to thepoly-alcohol could be achieved in the same manner as the chargedside-groups are coupled to the poly-alcohol. The binding of thefunctional group must not allow release of the functional group in vivo.

The compounds according to the invention can be administered alone, orin conjunction with a pharmaceutically acceptable carrier.

EXAMPLES

This invention will now be described in greater detail by reference tothe following non-limiting examples in which:

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an example of a compound according to the invention whereinthe amino acid lysine is coupled to dextran, according to the invention.

EXAMPLE 1

Synthesis of Tc-99m-lysine-dextran according to the invention.Activation of the hydroxyl groups of dextran. 20 mg of dextran (mean mw40000 Dalton, Pharmacia-Amersham Biotech, Sweden) is mixed with 12 mgsodium periodate in a total volume of 1mL 0.15 M sodium acetate, pH 5.5and incubated during stirring at room-temperature for 5 h. The polymeris then purified on a disposable Sephadex®-G25 (Pharmacia-AmershamBiotech, Sweden) gel filtration column.

Coupling of lysine to activated dextran. 20 mg of the activated dextranis mixed with a large molar excess of lysine (28 mg lysine), in 2.1 mLof 0.1M NaHCO₃ and 4mg of sodium cyanoborohydride is added. The mixtureis incubated during stirring at room temperature for ˜15 h. Afterincubation the compound is purified on a PD 10 column and the buffer isexchanged to 0.1M sodium acetate. This results in the coupling of alysine to approximately 15-30% of the glucose residues of dextran.

Labeling of lysine-dextran with technetium (Tc-99m). Labeling oflysine-dextran with Tc-99m is performed according to the method of Henzeet al. (9), i.e., 50 μg SnCl₂ (10 μL of konc. 5 g/L, in 99% ethanol) isadded to 50 μg lysine-dextran in 1 mL 0.1M sodium acetate. Then ˜100 MBqsodium pertechnetate (˜100 μL) is added and the reaction mixture isincubated for 15 minutes at room temperature. The solution is purifiedon a Sephadex®-G25 PD10 column (Pharmacia-Amersham Biotech, Sweden),sterile filtrated, and dilute in 150-200 ml saline. After labeling thecompound should be used directly. All solutions above are degassed. Theresulting Tc-99m lysine dextran contained 1 MBq per μg.

Example 2

This example serves to demonstrate that Tc-99m-lysine dextran accordingto the invention is selectively accumulated in tumor tissue.

Nine patients having epidermal growth factor positive superficialtransitional cell bladder carcinoma were studied. Tc-99m-dextran withdifferent charges and containing the same total amount of radio-activitywere instilled in the bladder of these patients. The instilled polymerswere Tc-99m-epidermal growth factor (EGF)-dextran, neutral dextran, i.e.Tc-99m-dextran, anionic dextran, i.e. Tc-99m-taurin dextran, andcationic dextran, i.e. Tc-99m-lysine dextran (according to Example 1).

200 mL of the polymers, of the concentration 0.25 μg/mL in saline, wereinstilled through an urethral catether and kept in the bladder for 30minutes and were then carefully washed out with approximately 400 mLsaline. The patients were then operated on and samples were taken fromnormal bladder tissue and bladder tumor tissue, respectively. Thesamples were weighed and counted in gamma counter and the radioactiveuptake was calculated as cpm/g tissue. The target to non-target (ttn)ratio was calculated. Results (mean values) are shown in Table 1.

TABLE 1 number of patients polymer radioactive uptake, ttn studiedTc-99m-EGF-dextran  700:1 4 Tc-99m-dextran  90:1 1 Tc-99m-taurin dextran  2:1 1 Tc-99m-lysine dextran 2050:1 2

The Tc-99m-lysine-dextran according to the invention resulted in thehighest tumor uptake of radioactivity, i.e. the highest target tonon-target ratio. Tc-99m-EGF-dextran serves as a comparison with priorart techniques, i.e. targeting to tumors by antibodies directed toproteins that normally are over-expressed in tumors. However, theconditions in this study were unfavorable for the interaction betweenthe Tc-99m-EGF-dextran and EGF-receptors expressed by tumors, hence thelarge ttn ratio was interpreted as charge dependent through charged sidegroups of Tc-99m-EGF-dextran.

REFERENCES

1. Hurwitz E. Specific and nonspecific macromolecule-drug conjugates forthe improvement of cancer chemotherapy. Biopolym. 1983; 22, 557-567.

2. Maeda H, Seymour L W, Miyamoto Y. Conjugates of anti-cancer agentsand polymers: Advantages of macromolecular therapeutics in vivo.Bioconj. Chem. 1992; 3, 5, 353-363.

3. Rogers K E, Carr B I, Tökes Z. Cell surface-mediated cytotoxicity ofpolymer-bound adriamycin against drug-resistant hepatocytes. Cancer Res.1983; 43, 2741-2748.

4. Schacht E, Vermeersch E et al. Synthyesis and characterization ofsome modified polysaccarides containing drug moieties. J. ControlledRelease. 1985; 2, 245-256.

5. Seymour L W. Passive tumor targeting of soluble macromolecules anddrug conjugates. Critical Rev. Therap. Drug Carrier Syst. 1992; 9,135-187.

6. Sezaki H, Hashida H et al. Macromolecule-drug conjugates in targetedcancer chemotherapy. Biodegradable polymers in controlled drug delivery.Critical Rev. Therap. Drug. Carrier Syst. 1984; 1,1, 1-37.

7. Comper W D and Laurent, T C. Physiological Function of ConnectiveTissue Polysaccarides. Physiological Reviews 58:255-315, 1978.

8. Grodzinsky A J. Electromechanical and Physiochemical Properties ofConnective Tissue. CRC Critical Reviews in Biomedical Engineering9:133-199.

9. Henze E and Robinson G D. Tc-99m dextran: A new blood-pool labelingagent for radionucllide angiocardiography. J. Nucl. Med. 23:348-353,1982

10. Foster, R L. Preparation and properties of a soloubletrypsin-dextran conjugate. Expertia 31:772-773, 1975.

11. Tabata et al. Drug Delivery 4:213-221, 1997.

12. Syrigos et al. Acta Oncologia Vol 38, No 3, pp.379-382, 1999

What is claimed is:
 1. A method for treating charged tumors by an ionexchange reaction comprising administering to a patient in need thereofan effective amount of a composition comprising dextran having a chargeopposite that of said charged tumor, in which the charge of said dextranhas been modified by covalent bonding of charged groups.
 2. The methodaccording to claim 1 wherein the composition is administered by a methodselected from the group consisting of local, intratumoral, andintracavitary.
 3. The method according to claim 1 wherein the chargedgroups are selected from the group consisting of amino acids withcharged side chains and derivatives thereof.
 4. The method according toclaim 3 wherein the amino acids are selected from the group consistingof ornithine, lysine, taurine, arginine, histidine, glutamic acid, andaspartic acid.
 5. The method according to claim 1 wherein the chargedgroups are bound to activated hydroxyl groups of dextran.
 6. The methodaccording to claim 1 wherein a charged group is bound to 15-30% of theglucose residues or dextran.
 7. The method according to claim 1 whereinthe dextran contains functional groups.
 8. The method according to claim7 wherein the functional groups are bound to the activated hydroxylgroups of dextran.
 9. The method according to claim 7 wherein thefunctional groups are selected from the group consisting ofradionuclides.
 10. The method according to claim 1 wherein the dextranhas a molecular weight of 10³ to 10⁶ daltons.
 11. The method accordingto claim 1 wherein the composition is localized to the tumor at a targetto non-target ratio of at least 600:1.
 12. The method according to claim1 wherein the charge of said dextran is adapted to the charge of thetumor to be treated, thereby increasing the target to non-target ratioof the compound to the tumor.
 13. The method according to claim 1wherein the tumor is a bladder carcinoma tumor.
 14. A method fortreating charged tumors by an ion exchange reaction comprisingadministering to a patient in need thereof an effective amount of acomposition comprising dextran, wherein the charge of the dextran hasbeen modified through covalent bonding of cationic groups and whereinthe charged tumor is an anionic tumor.
 15. The method according to claim14 wherein the composition is administered by a method selected from thegroup consisting of local, intratumoral, and intracavitary.
 16. Themethod according to claim 14 wherein the charged groups are bound toactivated hydroxyl groups of dextran.
 17. The method according to claim14 wherein a charged group is bound to 15-30% of the glucose residues ordextran.
 18. The method according to claim 14 wherein the dextran has amolecular weight of 10³ to 10⁶ daltons.
 19. The method according toclaim 14 wherein the composition is localized to the tumor at a targetto non-target ratio of at least 600:1.
 20. The method according to claim14 wherein the charge of said dextran is adapted to the charge of thetumor to be treated, thereby increasing the target to non-target ratioof the compound to the tumor.
 21. The method according to claim 14wherein the tumor is a bladder carcinoma tumor.